1. Field of the Invention
This invention relates to chemochromic sensor films for use in rapidly screening isolate organisms capable of producing hydrogen.
2. Description of the Prior Art
Light-induced biological hydrogen production represents a potentially cost-effective system for the production of renewable non-polluting energy. Photobiological hydrogen production is catalyzed by nitrogenase and hydrogenase enzyme systems that are present in bacteria, photosynthetic bacteria, cyanobacteria, and green algae. Green algae, such as Chlamydomonas reinhardtii, can photoevolve hydrogen only through an inducible, reversible hydrogenase enzyme using water as the electron source. While hydrogenase-catalyzed hydrogen photo-production has potential as an efficient energy source, one of the major obstacles which currently limits any commercial application of this process is the deactivation of the hydrogenase enzyme in the presence of oxygen, produced in the water-splitting process of photosynthesis. Thus, it is desirable to isolate and select for bacterial, or algal mutant organisms which exhibit an oxygen-tolerant hydrogen production phenotypic response, in order to further the commercial production of light-induced hydrogen.
Two recent methods have been used in order to select for C. reinhardtii mutants which exhibit the oxygen tolerant hydrogen production phenotypic response. These methods are based on either the hydrogen production or hydrogen uptake activity of the reversible hydrogenase enzyme. The first approach is dependent on the ability of algal cells to produce hydrogen in competition with a drug that, when reduced, releases products toxic to the cells. Hydrogen production selective pressure is applied in the presence of increasing oxygen-stress to enrich for oxygen-tolerant organisms. The second approach is based on algal cell growth, using hydrogen as an electron source for carbon dioxide fixation. The addition of oxygen during the hydrogen uptake selection is then used to select for oxygen-tolerant organisms.
The evolution of hydrogen by an organism has been assayed amperometriclly. The amperometric determination uses a Clark type electrode that can be biased versus Ag/AgCl to determine either the hydrogen or oxygen concentration present in an assay chamber. Algal cells are induced under anaerobic conditions prior to the measurements. The algae are then anaerobically injected into an assay chamber containing an assay buffer (50 mM MOPS, pH 6.8), which is pre-adjusted to different initial concentrations of oxygen. The cells are then incubated in the dark and illuminated with a saturating heat-filtered incandescent light to induce hydrogen evolution. Initial hydrogen evolution rates are derived from the initial slopes of each curve, and gas concentrations are corrected for the decrease in aqueous solution solubility. A gas chromatograph can also be used to assay for hydrogen production capacity. However, these assays for oxygen-tolerant hydrogen photo-production are very costly, time consuming, and have been the prime rate limiting factor in the rapid identification and selection of more desirable mutant organisms.
Hunter, in U.S. Pat. No. 5,668,301 discloses a hydrogen sensitive metal alloy that contains palladium and titanium to provide a larger change in electrical resistance when exposed to the presence of hydrogen. The alloy is deposited on a substrate and a thin film is connected across electrical circuitry to provide a sensor device that can be used for improved sensitivity and accuracy of hydrogen detection.
U.S. Pat. No. 5,367,283 issued to Lauf et al., discloses a thin-film hydrogen sensor element comprised of an essentially inert, electrically-insulating substrate having a thin-film metallization deposited thereon which forms at least two resistors on the substrate. The metallization comprises a layer of Pd or a Pd alloy for sensing hydrogen and an underlying intermediate metal layer for providing enhanced adhesion of the metallization to the substrate. The difference in electrical resistances of the covered resistor and uncovered resistor is related to the hydrogen concentration in a gas to which the sensor element is exposed.
U.S. Pat. No. 4,324,761, issued to Harris, discloses a hydrogen detector comprised of a substrate supporting a electrically conducting base metal film, and upper electrically conducting diffusion barrier metal film, a polycrystalline film of titanium dioxide sandwiched between the base and diffusion barrier films, the polycrystalline titanium dioxide film electrically insulates the base film from the diffusion barrier film, the base film, being in electrical contact with the titanium dioxide film, an insulating layer electrically insulating the titanium dioxide film from the diffusion barrier film except for a predetermined surface portion thereof in electrical contact with the diffusion barrier film; wherein the predetermined electrically contacting portion is sufficiently large to produce a measurable electrical conductance, an electrically conducting or non-conducting catalytic top film of metal able to dissociate hydrogen into its atomic form in electrical contact with the diffusion barrier film and a least coextensive with the barrier film throughout the predetermined electrically contacting portion, as can best be seen in the cross-sectional view of FIG. 2.
U.S. Pat. No. 4,324,760 to Harris, disclosed a hydrogen detector having a substrate supporting an electrically conducting base metal film, an electrically conducting top film of metal able to dissociate hydrogen into atomic form, a polycrystalline film of titanium dioxide sandwiched between the base and top films, wherein the polycrystalline titanium dioxide film electrically insulates the base film from the top film, the base film being in electrical contact with the titanium dioxide film, an insulating layer electrically insulating the titanium dioxide film from the top film except for a predetermined surface portion thereof in electrical contact with the top film, and wherein the predetermined electrically contacting portion is sufficiently large to produce a measurable electrical conductance that varies with the concentration of hydrogen in the atmosphere surrounding it.
Each of the foregoing patents issued to Hunter, Lauf, et al., and Harris disclose electrical devices, which generate an electrical signal, determinative of the presence of hydrogen. However, these devices do not spatially resolve the point where the gas in produced in relation to the sample surface. Therefore, these devices would not be useful to discriminate the specific location of a colony which produces hydrogen gas where the sample, to be screened, consists of many colonies of the organism.
U.S. Pat. No. 3,567,383 issued to Langley et al. discloses a detector for hydrogen having as its sensing device a thin film comprised of palladium or platinum oxide, which oxide on contact with hydrogen reduces to the corresponding metal. The differences in properties, electrical or optical, of the oxide and metal film are used to detect the presence of hydrogen. While this patent provides an optical means for detecting hydrogen which could provide spatial resolution it does not provide the sensitivity (0.02% hydrogen concentration), rapid response rate (a few seconds), and economy in manufacture which are desirable for rapidly screening colonies of hydrogen-producing organisms on a substrate.
The development of a rapid screening method and device for the detection of hydrogen-producing mutants would greatly enhance the analysis of the survivors of selective pressure methods, in the rapid isolation of desirable mutant organisms. Furthermore, a rapid screening would assist in the analysis of mutants derived through a molecular biological approach to further the oxygen-tolerance of the hydrogenase enzyme.
Therefore, what is needed, is a rapid screening method for the oxygen-tolerant, light-induced hydrogen phenotype in mutant organisms.